1. Field of the Invention
The present invention is directed to compositions and methods for increasing, and detecting the fluorescence of a molecule, in particular, compositions and methods for increasing the intrinsic fluorescence of a biomolecule. This invention also is directed to methods and compositions for the detection of biomolecules by increasing and detecting the fluorescence of biomolecules.
2. Description of Related Art
The use of fluorescence technology has greatly enhanced the ability to detect specific molecules leading to rapid advancements in diagnostics. For example, fluorescence detection is widely used in medical testing and DNA analysis because of the high degree of sensitivity obtained using fluorescent techniques. Small numbers of molecules can be detected using fluorescence technology. Typically, extrinsic fluorophores are added covalently or non-covalently to allow molecules that do not ordinarily fluoresce or do not fluoresce at previously commercially useful levels to be detected. Biomolecules such as DNA ordinarily do not fluoresce at detectable levels, and extrinsic fluorophores are added to DNA to facilitate the detection of DNA on gels (Benson et al. (1993) Nucleic Acids Res. 21, 5720-5726; Benson et al. (1995) Ananl. Biochem. 231, 247-255), in DNA sequencing (Smith et al. (1986) Nature 321, 674-679; Prober et al. (1987) Science 238, 336-343; Li et al. (1999) Bioconjugate Chem. 10, 241-245), in fluorescence in-situ hybridization (Denijn et al. (1992) APMIS 100, 669-681; Wiegant et la. (2000) Genome Res. 10, 861-865), and for reading of DNA arrays for gene expression (Lipshutz et al. (1999) Nat. Genet. Suppl. 0.1, 20-24; Ferea et al. (1999) Curr. Opin. Genet. Dev. 9, 715-722). Extrinsic fluorophores are used with DNA because DNA absorbs in the UV region so near 260 nm. The short absorption wavelength is now less of an obstacle because UV solid state lasers have become available. Nonetheless, the intrinsic fluorescence from DNA is of little practical usefulness because of the low quantum yields of 10−4 to 10−5 (Vigny et al. (1974) Photochem. Photobiol. 20, 345-349; Morgan et al. (1980) Photochem. Photobiol. 31, 101-113). Because the intrinsic emission from DNA, nucleotides and nucleic acid bases is very weak (Kneipp et al. (1999) Curr. Science 77, 915-924; Nie et al. (1997) Science 275, 1102-1106; Michaels et al. (1999) J. Am. Chem. Soc. 121, 9932-9939), it is difficult to observe the intrinsic fluorescence even with modern instrumentation (Gersten et al. (1985) Surface Science 158, 165-189; Lakowicz (2001) Anal. Biochem. 298, 1-24).
Some of the fluorescence techniques used to detect the presence of molecules include Resonance Energy Transfer (RET), immunofluorescent assays, and fluorescence in situ hybridization. Detection of the molecule of interest is generally limited by the properties of the fluorophore used. In some cases, labeling a biomolecule with an extrinsic fluorophore can alter the biological activity of the biomolecule potentially creating experimental artifacts. Problems with current fluorescent techniques stem in part from the low fluorescent intensities of commonly used fluorophores. Additionally, background fluorescence can be significant when using low wavelength excitation radiation required by some fluorophores or when large quantities of fluorophore are required.
DNA sequencing techniques using fluorescent dyes as markers have their maximum emission spectra in the visible range, the DNA is subject to irradiation in the visible spectra, and visible spectra detectors and light sources are used. Generally photomultipliers tubes are used for detection. As a result, these DNA sequencing techniques have several disadvantages including high costs resulting from the high cost of the lasers used to excite the fluorescent markers which typically emit in the visible region of light spectrum and the high noise to signal ratio due to the background interferences by biomolecules.
Thus, there is a need for compositions and methods for increasing the fluorescence intensity of biomolecules.
There is also a need for compositions and methods for increasing the intrinsic fluorescence of biomolecules.
Another need exists for compositions and methods for manipulating the fluorescence emission intensity of a biomolecule in response to an amount of exciting radiation.
Yet another need exists for methods and compositions for manipulating the radiative decay rate of biomolecules.